Red blood cells (RBCs) transport oxygen but accumulate oxidative damage over time, reducing function in vivo and during storage-critical for transfusions. To explore genetic influences on RBC resilience, we profiled proteins, metabolites, and lipids from fresh and stored RBCs obtained from 350 genetically diverse mice. Our analysis identified over 6,000 quantitative trait loci (QTL). Compared to other tissues, prevalence of trans genetic effects over cis reflects the absence of de novo protein synthesis in anucleated RBCs. QTL hotspots at Hbb, Hba, Mon1a, and storage-specific Steap3 linked ferroptosis to hemolysis. Proteasome components clustered at multiple loci, underscoring the importance of degrading oxidized proteins. Post-translational modifications (PTMs) mapped predominantly to hemoglobins, particularly cysteine residues. Loss of reactive C93 in humanized mice (HBB C93A) disrupted redox balance, affecting glutathione pools, protein glutathionylation, and redox PTMs. These findings highlight genetic regulation of RBC oxidation, with implications for transfusion biology and oxidative stress-dependent hemolytic disorders.

BACKGROUND:Storage of packed red blood cells (RBCs) for transfusion leads to biochemical and morphological changes, increasing hemolysis risk. Urate levels in blood bags at donation contribute to the molecular heterogeneity and hemolytic propensity of stored RBCs. However, studies to date have been underpowered to investigate at scale the contribution of donor demographics and genetics to the heterogeneity in urate levels across donations. STUDY DESIGN AND METHODS:Urate levels were measured in 13,091 RBC units from the REDS study. Characteristics tested included hemolysis parameters (spontaneous, osmotic, oxidative) at storage end and post-transfusion hemoglobin (Hb) increments in recipients. Donor demographics, urate levels, and genetic variants were analyzed for associations with these outcomes. RESULTS:Elevated urate levels were linked to male sex, older age, high BMI, and Asian descent. Units with high urate levels exhibited increased spontaneous and osmotic hemolysis, while oxidative hemolysis was unaffected. Genetic variants in SLC2A9 (V282I) and ABCG2 (Q141K) were strongly associated with elevated urate, particularly in Asian donors. Post-transfusion analyses revealed that units from female donors carrying these variants were associated with reduced Hb increments, with up to a 31% reduction in efficacy. This effect was not observed in male donors. DISCUSSION:RBC urate levels and genetic traits significantly impact storage quality and transfusion outcomes. These findings highlight the importance of donor molecular characteristics for optimizing transfusion strategies. Moreover, genetic and metabolic insights may inform donor recruitment efforts, providing health feedback to volunteers while ensuring effective transfusion products.

ABSTRACT:Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. However, the genetic underpinnings of RBC metabolic heterogeneity and extravascular hemolysis at population scale are incompletely understood. On the basis of the breeding of 8 founder strains with extreme genetic diversity, the Jackson Laboratory diversity outbred population can capture the impact of genetic heterogeneity in like manner to population-based studies. RBCs from 350 outbred mice, either fresh or stored for 7 days, were tested for posttransfusion recovery, as well as metabolomics and lipidomics analyses. Metabolite and lipid quantitative trait loci (QTL) mapped >400 gene-metabolite associations, which we collated into an online interactive portal. Relevant to RBC storage, we identified a QTL hotspot on chromosome 1, mapping on the region coding for the ferrireductase 6-transmembrane epithelial antigen of the prostate 3 (Steap3), a transcriptional target to p53. Steap3 regulated posttransfusion recovery, contributing to a ferroptosis-like process of lipid peroxidation, as validated via genetic manipulation in mice. Translational validation of murine findings in humans, STEAP3 polymorphisms were associated with RBC iron content, lipid peroxidation, and in vitro hemolysis in 13 091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. QTL analyses in humans identified a network of gene products (fatty acid desaturases 1 and 2, epoxide hydrolase 2, lysophosphatidylcholine acetyl-transferase 3, solute carrier family 22 member 16, glucose 6-phosphate dehydrogenase, very long chain fatty acid elongase, and phospholipase A2 group VI) associated with altered levels of oxylipins. These polymorphisms were prevalent in donors of African descent and were linked to allele frequency of hemolysis-linked polymorphisms for Steap3 or p53. These genetic variants were also associated with lower hemoglobin increments in thousands of single-unit transfusion recipients from the vein-to-vein database.

Aging results in a progressive decline in physiological function due to the deterioration of essential biological processes. While proteomics offers insights into aging mechanisms, prior studies are limited in proteome coverage and lifespan range. To address this, we integrate the Orbitrap Astral Mass Spectrometer with the multiplex tandem mass tag (TMT) technology to profile the proteomes of cortex, hippocampus, striatum and kidney in the C57BL/6JN mice, quantifying 8,954 to 9,376 proteins per tissue (12,749 total). Samples spanned both sexes and three age groups (3, 12, and 20 months), representing early to late adulthood. To improve TMT quantitation accuracy, we develop a peptide-spectrum match-based filtering strategy that leverages resolution and signal-to-noise thresholds. Our analysis uncovers distinct tissue-specific patterns of protein abundance, with age and sex differences in the kidney and primarily age-related changes in brain tissues. We also identify both linear and non-linear proteomic trajectories with age, revealing complex protein dynamics over the adult lifespan. Integrating our findings with early developmental proteomic data from brain tissues highlights further divergent age-related trajectories, particularly in synaptic proteins. This study provides a robust data analysis workflow for Orbitrap Astral-based TMT analysis and expands the proteomic understanding of aging across tissues, ages, and sexes.

The health risks that arise from environmental exposures vary widely within and across human populations, and these differences are largely determined by genetic variation and gene-by-environment (gene-environment) interactions. However, risk assessment in laboratory mice typically involves isogenic strains and therefore, does not account for these known genetic effects. In this context, genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening because they provide a way to introduce genetic variation in risk assessment without increasing animal use. Cell lines from genetic reference populations of laboratory mice offer genetic diversity, power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Increasing global life expectancy motivates investigations of molecular mechanisms of aging and age-related diseases. This study examines age-associated changes in red blood cells (RBCs), the most numerous host cell in humans. Four cohorts, including healthy individuals and patients with sickle cell disease, were analyzed to define age-dependent changes in RBC metabolism. Over 15,700 specimens from 13,757 humans were examined, a major expansion over previous studies of RBCs in aging. Multi-omics approaches identified chronological age-related alterations in the arginine pathway with increased arginine utilization in RBCs from older individuals. These changes were consistent across healthy and sickle cell disease cohorts and were influenced by genetic variation, sex, and body mass index. Integrating multi-omics data and metabolite quantitative trait loci (mQTL) in humans and 525 diversity outbred mice functionally linked metabolism of arginine during RBC storage to increased vesiculation-a hallmark of RBC aging-and lower post-transfusion hemoglobin increments. Thus, arginine metabolism is a biomarker of RBC and organismal aging, suggesting potential new targets for addressing sequelae of aging.

There is considerable variability in post-transfusion recovery (PTR) of stored red blood cells (RBCs). Both ferroptosis (iron oxidation and lipid peroxidation) and also eryptosis (programmed cell death involving dysregulated calcium and potassium) have been implicated in poor PTRs. However, the genetic basis for donor variability of PTR remains unclear. Large human genome-wide association studies (GWAS) using PTR as a phenotype (e.g., 51-chromium labeled RBCs) are not feasible. In such a case, a genetically diverse population of outbred mice (DO mice) is a powerful instrument of discovery for genetic mapping. Peripheral blood was collected into CPDA-1 from 350 DO donor mice, stored for 7 days, and transfused into GFP+ recipient mice. PTR was determined by enumerating circulating GFP-RBCs 24-h post-transfusion. Each donor was genotyped using a dense array of 143,000 markers and had a metabolome and lipidome generated on fresh and stored RBCs using liquid-chromatography mass spectrometry. Quantitative trait loci (QTL) analysis was carried out to map genetic loci associated with PTR, each metabolite, and each lipid. QTLs were integrated together to construct a genetic co-regulatory network and identify QTL hubs. Each of 21 oxylipids associated with poor PTR mapped a QTL with genome-wide adjusted p-values (adjP) < .05 on Chromosome 1 near Steap3, which is a ferrireductase directly involved in ferroptosis. Five Steap3-associated eicosanoids also had a secondary QTL at a region of Chromosome 16 containing Kcne1, a potassium voltage-gated channel known to be involved in eryptosis (adjP < .05). QTL mapping on metabolites that did not change with storage identified an association between oxidized glutathione levels and a hemoglobin beta (Hbb) variant with an extra cysteine predicted to provide a free sulfhydryl (adjP < 2.2e-16). Additional hubs for nucleotide metabolism mapped to CD38 on Chromosome 5 and Mon1a on Chromosome 9. These data identify genes involved with ferroptosis (Steap3) and eryptosis (Kcne1) as associated with PTR and metabolic perturbations of stored murine RBCs. The Steap3 finding confirms and extends previous work identifying increased Steap3 as a driver of lipid peroxidation and poor PTR in mice. In addition to identifying key genetic associations, these data also support the general concepts that ferroptosis and/or eryptosis are important biologies in RBC storage. Finally, generated data are a powerful resource for studying genetic control of metabolite and lipid levels in RBCs, to validate findings across studies and generate hypotheses for future investigations.

BackgroundGenetic variation influences both chromatin accessibility, assessed in chromatin accessibility quantitative trait loci (caQTL) studies, and gene expression, assessed in expression QTL (eQTL) studies. Genetic variants can impact either nearby genes (cis-eQTLs) or distal genes (trans-eQTLs). Colocalization between caQTL and eQTL, or cis- and trans-eQTLs suggests that they share causal variants. However, pairwise colocalization between these molecular QTLs does not guarantee a causal relationship. Mediation analysis can be applied to assess the evidence supporting causality versus independence between molecular QTLs. Given that the function of QTLs can be cell-type-specific, we performed mediation analyses to find epigenetic and distal regulatory causal pathways for genes within two major cell types of the developing human cortex, progenitors and neurons.ResultsWe find that the expression of 168 and 38 genes is mediated by chromatin accessibility in progenitors and neurons, respectively. We also find that the expression of 11 and 12 downstream genes is mediated by upstream genes in progenitors and neurons. Moreover, we discover that a genetic locus associated with inter-individual differences in brain structure shows evidence for mediation of SLC26A7 through chromatin accessibility, identifying molecular mechanisms of a common variant association to a brain trait.ConclusionsIn this study, we identify cell-type-specific causal gene regulatory networks whereby the impacts of variants on gene expression were mediated by chromatin accessibility or distal gene expression. Identification of these causal paths will enable identifying and prioritizing actionable regulatory targets perturbing these key processes during neurodevelopment.

Maintenance of protein homeostasis degrades with age, contributing to aging-related decline and disease. Previous studies have primarily surveyed transcriptional aging changes. To define the effects of age directly at the protein level, we perform discovery-based proteomics in 10 tissues from 20 C57BL/6J mice, representing both sexes at adult and late midlife ages (8 and 18 months). Consistent with previous studies, age-related changes in protein abundance often have no corresponding transcriptional change. Aging results in increases in immune proteins across all tissues, consistent with a global pattern of immune infiltration with age. Our protein-centric data reveal tissue-specific aging changes with functional consequences, including altered endoplasmic reticulum and protein trafficking in the spleen. We further observe changes in the stoichiometry of protein complexes with important roles in protein homeostasis, including the CCT/TriC complex and large ribosomal subunit. These data provide a foundation for understanding how proteins contribute to systemic aging across tissues.

Background: Red blood cells (RBCs) have a complex biology that allows regulation of metabolism, cellular rigidity, ion exchange, pH, and molecular communication with the vasculature. A central regulator of these biologies are macrocomplexes of integral membrane proteins that are both linked to cytoskeletal proteins and also differentially bind to metabolic enzymes based upon oxygenation status of the RBC. RBC surface staining, co-immunoprecipitation, and cross-linking proteomic approaches have generated a model of the macrocomplex encompassing Band 3, Rh, RhAG, CD47, glycophorin A, glycophorin B, and LW. TER-119 is a monoclonal antibody with exquisite specificity for the erythroid lineage in mice. However, the molecular target of TER-119 has remained unknown. TER-119 immunoprecipitates 4 specific protein bands, two of which are glycophorin A (GYPA) monomers or homodimers respectively; however, TER-119 is non-reactive to GYPA when assayed through Western blots and flow cytometry with erythroid cell lines expressing GYPA. These data led the originators of TER-119 to conclude it binds to a GYPA associated protein, but not GYPA itself. In contrast, others have concluded that TER-119 binds GYPA directly, and have inferred GYPA biology based upon TER-119 reactivity. Methods: TER-119 reactivity with peripheral blood RBCs from mice or from transfected HEK cells was determined by flow cytometry. Mice were genotyped using a dense array of 143,000 markers. Quantitative trait loci (QTL) analysis was carried out using TER-119 staining as a trait. cDNA for the open reading frames of the variants of interest in candidate genes or GYPA were cloned into eukaryotic expression vectors drive by the CMV promoter. HEK cells were transfected with expression vectors, in combination with a GFP expression plasmid. Cells were stained with TER-119 48 hours post transfection and analyzed by flow cytometry, gating on GFP positive cells to isolate the transfected population. Hematopoietic lineage expression of mRNA was obtained from the BloodSpot database. Results: The Diversity Outbred mice (DO mice) are an outbred population descended from 8 parental strains (A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). While analyzing RBC biology phenotypes in DO mice, using TER-119 as a marker of RBCs, we serendipitously observed TER-119 non-reactivity in peripheral blood of 5 out of 550 DO mice. QTL analysis using TER-119 reactivity as a trait identified a highly significant QTL (genome-wide adjusted p-value <1.11e-16) driven by alleles specific to WSB/EiJ mice. Analysis of each of the parental strains confirmed that only peripheral blood from WSB/EiJ mice was non-reactive with TER-119. The QTL region contained 4 genes with missense mutations specific to the WSB/EiJ strain (HTR3a, HTR3b, NXPE2, and NXPE4). TER-119 was weakly reactive with HEK cells transfected with NXPE2 (but not the other genes). However, TER-119 became strongly reactive with HEK cells co-transfected with NXPE2 and GYPA (but not the other genes co-transfected with GYPA). There is no GYPA coding variation present in 8 DO parental strains. NXPE2 mRNA is highly expressed in the murine erythroid lineage with lower expression in B cells and granulocytes. Discussion: These data demonstrate that TER-119 reactivity is conferred by co-expression of NXPE2 and GYPA. NXPE2 is a single pass transmembrane protein and the weak reactivity of TER-119 with NXPE2 alone suggests that TER-119 recognizes an epitope on NXPE2 that is allosterically enhanced by complexing with GYPA. However, other binding scenarios cannot be ruled out. These data are important for three reasons. First, they identify NXPE2 as a hitherto unrecognized surface protein specific to the erythroid lineage in mice. Second, they raise the possibility that the models of the macrocomplex are incomplete. Third, these findings may allow re-interpretation of existing data that assumed TER-119 recognized GYPA. Additional studies are required to determine what function (if any) NXPE2 plays in erythropoiesis in mice and whether this function translates to human NXPE2 or a homologous gene.

Glycolysis is a central metabolic pathway in health and disease,1 an essential one in mature red blood cells (RBCs), which rely on the Embden-Meyerhof-Parnas pathway, i.e., glycolysis, as the sole source of energy generation.2 During aging in vivo and in vitro under blood bank conditions,3 the adenosine triphosphate (ATP) generated via glycolysis in mitochondria-devoid mature RBCs is essential for the modulation of oxygen kinetics,4 deformability2 and, ultimately, intra- and extra-vascular hemolysis.3 Here we draw upon a cohort of 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study (REDS) to identify biological and genetic traits associated with heterogeneity in glycolytic phenotypes. Age, sex and ethnicity influenced glycolysis, with additional effects due to genetic polymorphisms in regions coding for rate-limiting enzymes of glycolysis,1 phosphofructokinase 1 (PFKP) and hexokinase 1 (HK1), and for the ADP-ribosyl cyclase CD38. The gene-metabolite associations were validated by testing glycolysis in fresh and stored RBCs from 350 Diversity Outbred mice.5 These findings were further corroborated via multi-omics analyses of 1,929 samples at storage day 10, 23 and 42 in 643 RBC units from donors who were selected amongst the index cohort based on their extreme hemolytic propensity. Finally, we show that glycolysis, ATP levels, breakdown and deamination into hypoxanthine are associated with increased hemolytic propensity and vesiculation ex vivo, as well as with extravascular hemolysis in vivo, both in healthy autologous transfusion recipients from the Donor Iron Deficiency Study and in 4,700 heterologous critically ill patients receiving blood products from the REDS donors. This work advances our understanding of genetic and non-genetic factors that regulate glycolysis in mature RBCs, a simplified model of mammalian cell metabolism,6 a cell type where dysregulated glycolysis holds broader implications in hemolytic disorders, offering potential avenues for novel therapeutic interventions and transfusion strategies. Graphical abstract

Mediation analysis is used in genetic mapping studies to identify candidate gene mediators of quantitative trait loci (QTL). We consider genetic mediation analysis of triplets—sets of three variables consisting of a target trait, the genotype at a QTL for the target trait, and a candidate mediator that is the abundance of a transcript or protein whose coding gene co-locates with the QTL. We show that, in the presence of measurement error, mediation analysis can infer partial mediation even in the absence of a causal relationship between the candidate mediator and the target. We describe a measurement error model and a corresponding latent variable model with estimable parameters that are combinations of the causal effects and measurement errors across all three variables. The relative magnitudes of the latent variable correlations determine whether or not mediation analysis will tend to infer the correct causal relationship in large samples. We examine case studies that illustrate the common failure modes of genetic mediation analysis and demonstrate how to evaluate the effects of measurement error. While genetic mediation analysis is a powerful tool for identifying candidate genes, we recommend caution when interpreting mediation analysis findings.

Genetic background drives phenotypic variability in pluripotent stem cells (PSCs). Most studies to date have used transcript abundance as the primary molecular readout of cell state in PSCs. We performed a comprehensive proteogenomics analysis of 190 genetically diverse mouse embryonic stem cell (mESC) lines. The quantitative proteome is highly variable across lines, and we identified pluripotency-associated pathways that were differentially activated in the proteomics data that were not evident in transcriptome data from the same lines. Integration of protein abundance to transcript levels and chromatin accessibility revealed broad co-variation across molecular layers as well as shared and unique drivers of quantitative variation in pluripotency-associated pathways. Quantitative trait locus (QTL) mapping localized the drivers of these multi-omic signatures to genomic hotspots. This study reveals post-transcriptional mechanisms and genetic interactions that underlie quantitative variability in the pluripotent proteome and provides a regulatory map for mESCs that can provide a basis for future mechanistic studies.

TransfusionVolume 63, Issue S5 p. 46A-47A SUPPLEMENT ARTICLE OA3-AM23-MN-19 | Immunodominance to Naturally Occurring RBC Alloantigens is Controlled by Non-Genetic Factors in Mice A. Jash, A. Jash University of VirginiaSearch for more papers by this authorJ. Collins, J. Collins University of VirginiaSearch for more papers by this authorA. Hay, A. Hay University of VirginiaSearch for more papers by this authorG. Keele, G. Keele Jackson LabsSearch for more papers by this authorG. Churchill, G. Churchill Jackson LabsSearch for more papers by this authorK. Hudson, K. Hudson Columbia University Irving Medical CenterSearch for more papers by this authorC. Luckey, C. Luckey University of VirginiaSearch for more papers by this authorJ. Zimring, J. Zimring University of VirginiaSearch for more papers by this author A. Jash, A. Jash University of VirginiaSearch for more papers by this authorJ. Collins, J. Collins University of VirginiaSearch for more papers by this authorA. Hay, A. Hay University of VirginiaSearch for more papers by this authorG. Keele, G. Keele Jackson LabsSearch for more papers by this authorG. Churchill, G. Churchill Jackson LabsSearch for more papers by this authorK. Hudson, K. Hudson Columbia University Irving Medical CenterSearch for more papers by this authorC. Luckey, C. Luckey University of VirginiaSearch for more papers by this authorJ. Zimring, J. Zimring University of VirginiaSearch for more papers by this author First published: 12 October 2023 https://doi.org/10.1111/trf.46_17554Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume63, IssueS5October 2023Pages 46A-47A RelatedInformation

Red blood cell (RBC) storage in the blood bank induces a series of biochemical and morphological alterations, collectively denoted as the storage lesion(s). Membrane damage by lipid peroxidation is a hallmark of the storage lesion, a process thought to be triggered by oxidant stress. To identify the genetics underlying this phenomenon, we leveraged a novel murine system, the Jackson lab Diversity Outbred (JDO) mouse colony, which was obtained by extensively cross-breeding 8 founder mouse strains with extreme genetically heterogeneity ( Figure 1.A). A total of 350 fresh and stored (up to 7 days) murine RBC samples were tested by mass spectrometry-based metabolomics and lipidomics. At the end of storage, post-transfusion recovery (PTR), which measures intra- and extravascular hemolysis and is a gold standard for determining RBC storage quality, was determined by quantifying the percentage of stored RBCs that were still circulating 24h after their transfusion into Ubi-GFP+ recipient mice ( Figure 1.A). Genome wide-association studies identified a strong association between PTR and a polymorphic region on chromosome 1 ( Figure 1.A), which encodes STEAP3, a ferrireductase. This region was associated with altered metabolism of stored RBCs ( Figure 1.A), especially an elevation of lipid peroxidation products (hive plot in Figure 1.A). Mechanistically, these data support a role for STEAP3 in promoting Fenton and Haber-Weiss chemistry in iron-loaded RBCs. Specifically, ferrous iron participates in this chemistry, becoming oxidized to its ferric state with concomitant generation of hydroxyl or hydroperoxyl radicals; these in turn attack fatty acids (preferentially, poly- and highly-unsaturated fatty acids, such as octadecadienoic and eicosatetraenoic acid), thereby promoting lipid peroxidation (to hydroxyoctadecadienoic (HODE) and hydroxyeicosatetraenoic (HETE) acids, respectively). By reducing Fe 3+ to Fe 2+, STEAP3 can shift the balance of this reaction by increasing the availability of the reactant, thereby favoring the generation of product radicals; this process is analogous to ferroptosis, as described in other biological systems. To extend the relevance of these findings to humans, we performed metabolomics on end-of-storage (i.e., Day 42) packed RBCs from 13,091 human donors in the Recipient Epidemiology and Donor Evaluation Study (REDS RBC Omics; Figure 1.B), enrolled in four blood centers across the United States. We stratified donors based on oxylipin levels (e.g., acids - HETEs - Figure 1.B) and common STEAP3 polymorphisms (e.g., a representative non-synonymous coding single nucleotide polymorphism (SNP), rs17013371, was observed with ~10% prevalence in these donors), along with 37 other common SNPs observed in this study ( Figure 1.B). When correlating metabolic and hematological parameters to common STEAP3 non-synonymous coding SNPs, we found strong associations of such polymorphisms with RBC susceptibility to hemolysis (either osmotic or oxidative), lipid peroxidation, RBC numbers, and hemoglobin levels ( Figure 1.B). These associations held true for a subset of the original donors with extreme hemolytic propensity (n=643) who donated a second RBC unit that was again stored for 42 (i.e., the “recalled donor population”). In the initial (n=13,091) and recalled donor (n=643) populations, lipid peroxidation products, HETEs, and HODEs ranked as the top predictors of hemolysis and vesiculation. In summary, we identified a role for the STEAP3 ferrireductase in contributing to a ferroptosis-like phenotype of lipid peroxidation that is associated with increased susceptibility to hemolysis in vitro and in vivo in murine and human RBCs following storage under blood bank conditions. Figure 1 - A. Jackson Lab diversity outbred (JDO) mice were bred from 8 founder mouse strains with extreme genetic heterogeneity. A total of 350 fresh and stored murine red blood cells (RBCs) were tested for metabolomics, lipidomics, and post-transfusion recovery (PTR). A polymorphic region on chromosome 1, coding for the ferrireductase STEAP3 was associated with heterogeneous PTR and elevation in lipid peroxidation products. B. Metabolomics analyses of 13,091 donors from the REDS RBC Omics identified an association between the end of storage levels of oxylipins and common polymorphisms for STEAP3, both linked to an increased RBC susceptibility to hemolysis (either osmotic or oxidative.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.